In parallel with the "Jennerian" and modified "Jennerian" approach to vaccination against rotavirus diarrhea in which an oral live attenuated rotavirus vaccine is generated, we are pursuing other exploratory approaches to vaccination. A complete rotavirus particle consists of three capsid layers: an outer layer consisting of VP4 and VP7, an intermediate layer consisting of VP6 and an inner core consisting primarily of VP2. In this project, taking advantage of (i) the availability in our laboratory of various baculovirus recombinants expressing rotavirus proteins including VP2, VP4, VP6 and VP7 and (ii) properties of selected rotavirus structural proteins to self-assemble spontaneously and form virus-like particles (VLPs) upon co-expression in insect cells, we are pursuing the construction of recombinant rotavirus VLP subunit vaccines that can be delivered via various routes including intranasally. By co-infecting insect cells with recombinant baculoviruses expressing simian rotavirus SA11 VP2 and VP6 and human rotavirus AU32 VP7 (G9), we initially analyzed various factors involved in VLP assembly in an effort to optimize the expression system and enhance the yield of VLPs. Although double-layered VP2/6 VLP self-assembly was readily accomplished upon co-infection with VP2 and VP6 recombinant baculoviruses, self-assembly of triple-layered (VP2/6/7) VLPs appeared to be enhanced by infecting cells first with VP7 recombinant baculovirus followed approximately 6 hours later by infection with VP2 and VP6 recombinant baculoviruses.